LNK (SH2B3) Synergizes with TP53 in Suppressing B-Precursor Acute Lymphoblastic Leukemia through IL-7 Receptor Signaling

B-precursor acute lymphoblastic leukemia (B-ALL) is the leading cause of cancer-related deaths in children and commonly has a poor outcome in adults. Gene profiling and exome sequencing of high-risk ALLs led to the recent identification of the Philadelphia chromosome (Ph)-like ALL subtype. These leukemias have gene expression profiles similar to BCR-ABL1-positive (Ph⁺) ALL, but lack a BCR-ABL1 rearrangement, and often result in poor outcomes. Many Ph-like ALL-associated mutations identified to date are known or predicted to activate oncogenic cytokine receptor signaling pathways, particularly those associated with JAK. The lymphocyte adaptor protein LNK (also called SH2B3) has emerged as a powerful negative regulator of cytokine-mediated JAK2 signaling in hematopoietic stem cells (HSCs). Loss-of-function LNK mutations and deletions have recently been described in B-ALL, and germline loss of LNK contributes to pediatric ALL development. However, the mechanisms by which LNK alterations impact leukemogenesis remain poorly understood. Here we show that LNK synergizes with TP53 and INK4a in suppressing B-ALL development in mice. Tp53^-/-Lnk^-/- mice developed highly aggressive and transplantable B-ALL with 100% penetrance in contrast to T-lymphoma or sarcoma development observed in Tp53^-/- mice. Importantly, gene expression profiles of Tp53^-/-Lnk^-/- B-ALL blasts were similar to those of human Ph-like B-ALLs, validating the relevance of this model for preclinical studies. Tp53^-/-Lnk^-/- pro-B progenitors initiated B-ALL in the transplanted recipients, and Lnk loss-of-heterozygosity (LOH) was found exclusively in B-ALL cells from Tp53^-/-Lnk^+/- mice, but not in HSCs, common lymphoid progenitors, or cells of myeloid/T cell lineages, attesting that LNK is a bona fide tumor suppressor in the committed B-cell precursors. Mechanistically, we found that pre-leukemic Lnk^-/-Tp53^-/- pro-B progenitors were hypersensitive to IL-7 and showed markedly enhanced self-renewal ability in vitro and in vivo. Our genetic studies revealed that LNK controls normal B cell and B-ALL development independently of its effects on HSCs and specifically regulates pro-B cell homeostasis. A novel phosphoflow cytometry approach in freshly isolated BM cells that combines the surface marker B220, intracellular mu-heavy chain detection, and phospho-specific antibodies enabled separation of subpopulations of B progenitors with distinct IL-7 responsiveness. Using this approach, we demonstrated that LNK deficiency potentiated STAT5 activation in response to IL-7 in pre-leukemic pro-B cells. Of note, Lnk^-/-p53^-/- leukemic blasts with elevated pSTAT5, were less sensitive to JAK inhibitors than pre-leukemic B progenitors, in part due to constitutive activation of MAPK and AKT/mTOR pathways. Our results invoke the targeting of these pathways as novel therapeutic approaches in B-ALL. In sum, we have developed a novel B-ALL mouse model suitable for preclinical studies aimed at further deciphering the pathogenic mechanisms underlying this disease and exploring new therapeutic strategies.